BRPI0713389A2 - process for combined degreasing and washing of a starched fabric, and, composition - Google Patents

Abstract

PROCESS FOR COMBINED DESIGNING AND WASHING OF AN IRONED FABRIC AND COMPOSITION. The present invention relates to processes for combined degumming and cleaning of a starch-containing starch fabric or starch derivatives during fabric manufacture, which process comprises incubating said starch fabric in an aqueous treatment solution having a pH in the range of 1 and 7, aqueous treatment solution is comprising an acid amylase and at least one other acid enzyme facilitating said further processing steps. The present invention further relates to compositions used in said processes and the use of said composition.

Description

"PROCESS FOR COMBINED DESIGNING AND WASHING OF AN IRONED FABRIC AND COMPOSITION"

REFERENCE TO A LIST OF SEQUENCES

This request contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to combined degumming and cleaning processes employing acidic amylase and other enzymes such as cellulase, pectinase, lipase, xylanase, protease etc. during manufacture of new tissues.

BACKGROUND OF THE INVENTION

Fabric processing, such as cellulosic material, into ready-made garment material involves several steps: spinning the fiber into a yarn; construction of woven or interwoven wire cloth; and subsequent preparation, dyeing and finishing operations. The preparation process, which may involve degreasing (for woven goods), cleaning and bleaching, produces a fabric suitable for dyeing or finishing.

WO 2006/002034 (Novozymes) describes a simultaneous degumming and cleaning process comprising treating the tissue with an alkaline alpha amylase and an alkaline cleaning enzyme. Alkaline alpha-amylases are used as aids in degumming processes to facilitate the removal of starch-containing gum, which served as a protective coating on the yarns during weaving.

Complete removal of the gum coating after weaving is important to ensure optimum results in subsequent processes in which the fabric is generally cleaned, bleached, dyed and / or printed.

After the degumming step, it is often desirable to include a demineralization step in order to remove metal ions such as Mn2 +, Fe2 / Fe3 +, Cu2 + etc., which - if present in the fabric - may result in uneven bleaching in a further processing step or could even produce pinholes in the bleached fabric. Demineralization is typically performed by acid precipitation and typically involves the addition of acids such as acetic acid or sulfuric acid.

There is a need for improved processes for simultaneous degreasing combined with other fabric treatment steps such as combined degreasing and cleaning, combined degreasing and biopolishing, combined degreasing and abrasion and combined degreasing and carbonization etc.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to the provision of degumming processes of starched fabrics during manufacture of especially new fabrics under acidic conditions.

In one aspect, the present invention relates to a process for degumming and other combined tissue treatment steps of starch containing starch or starch derivatives during fabric manufacture, which process comprises incubating said starch fabric in a solution. Aqueous treatment solution having a pH in the range from 1 to 7, preferably from 1 to 5, especially from 1 to 4, an aqueous treatment solution comprising an acid amylase and at least one other acid enzyme facilitating said other one (s). ) tissue treatment step (s).

Preferably, said other acid enzymes (s), facilitating said other tissue treatment step (s), are acid cellulase, acid pectinase, acid lipase, acid xylanase and / or acid protease. More preferably, the enzyme (s) facilitating said other tissue treatment step (s) is acidic pectinase (s).

Preferably, the acid amylase is of bacterial or fungal origin, such as filamentous fungus origin.

Preferably, the acid amylase is derived from a strain of Aspergillus, preferably Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, or Aspergillus kawachii (SEQ ID NO: 37) or a strain of Rhizomucor, preferably Rhizomucor pusillus, or Merus a strain of Meripilus giganteus. Most preferably Aspergillus acid amylase is Aspergillus niger acid alpha amylase described in SEQ ID NO: 48, or a variant thereof.

Preferably, the bacterial acid amylase is derived from a strain of the genus Bacillus, preferably derived from a strain of Bacillus sp., More preferably a strain of Bacillus licheniformes, Bacillus amyloliquefaciens, Bacillus stearothermoptilus, Bacillus subtilis, or Bacillus Sp. Sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375, DSMZ 12648, DSMZ 12649, KSM AP1378, KSM K36 or KSM K38.

Hybrid alpha-amylase may also be used in the present invention. Preferably, the hybrid alpha-amylase could be amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glycosylase linker and SBD described as V039 in Table 5 of co-pending International Application no. PCT / US 05/46725.

Preferably, the acidic alpha-amylase is present at a concentration of 1 - 3,000 AFAU / kg tissue, preferably 10 - 1,000 AFAU / kg tissue, especially 100 - 500 AFAU / kg tissue or 1 - 3,000 AFAU / 1 tissue solution. treatment, preferably 10 - 1,000 AFAU / 1 treatment solution, especially 100 - 500 AFAU / 1 treatment solution.

Preferably, the alpha amylase is the hybrid alpha amylase shown in SEQ ID NO: 48, comprising a Rhizomucor pusillus alpha amylase catalytic domain (CD) having an A. niger carbohydrate binding domain (CBD).

There are usually three types of pectic enzymes: pectesterase, depolymerizing enzymes and protopectinase. Preferably said acid pectinase is an acid pectate lyase, an acid pectin lyase, an acid polygalacturonase and / or an acid lyase polygalacturonate. More preferably, the acid pectinase is Pectinex BEE XXL, Pectinex Ultra; Pectinex Yield Mash, Pectinex XXL, Pectinex Smash XXl or mixtures thereof.

Preferably, the acid pectinase is of the genus Aspergillus. Preferably, the acid pectinase may be added into the solution before, simultaneously or after the addition of acidic amylase.

Preferably, the process is carried out at a temperature in the range of 5 - 90 ° C, in particular 20 to 90 ° C. More preferably, the process is carried out at a temperature between 25 and 60 ° C for a suitable period of time, preferably between 2 and 24 hours.

Preferably, the fabric is made from natural or man-made fibers, cotton fabric, denim (resistant cotton fabric), flax, ramie, viscose, lyocell or cellulose acetate.

Preferably, the fabric is made of animal fibers, in particular silk or wool.

Preferably, the fabric is made of man-made or naturally-sourced polyester fibers such as poly (ethylene terephthalate) or poly (lactic acid) or nylon, acrylic or polyurethane fibers. The fabric is preferably a polyester-containing fabric or garment and consists essentially of 100% polyester. The polyester fabric is a polyester blend, such as a blend of polyester and cellulosic, including blends of polyester and cotton; a blend of polyester and cotton; a mixture of polyester and silk; a mixture of polyester and acrylic acid; a mixture of polyester and nylon; a mixture of polyester, nylon and polyurethane; a mixture of polyester and polyurethane, rayon (viscose), cellulose acetate and tencel.

In another aspect, the present invention relates to a composition comprising an acid amylase and an acid wash enzyme. Acid amylase is preferably derived from Aspergillus niger or Rhizomucor pusillus or mixtures thereof. The wash enzyme is preferably selected from the group consisting of acid cellulase, acid pectinase, acid lipase, acid xylanase and / or acid protease and mixtures thereof.

Preferably said acid pectinase is Pectinex® BE XXL, Pectinex® BE Color, Pectinex® Ultra; Pectinex ™ Ultra SP-L, Pectinex® Yield SVIash, Pectinex® XXL, Pectinex® Smash XXL, Pectinex® Smash and / or Pectinexw AR. Said acid pectinase is preferably derived from an Aspergillus strain. The composition further comprises stabilizer, surfactant, wetting agent, dispersing agents, sequestering agents and emulsifying agents, or a mixture thereof.

In the third aspect, the present invention relates to the use of the composition as described above for simultaneous degreasing and washing.

It has been found that when performing a simultaneous degumming and biolashing process of the invention as defined in the claims, no demineralization is required. Demineralization occurs simultaneously and / or after degumming and biolavaging of the starched tissue in the same treatment solution. Compared to traditional processes involving an acid degumming step and a demineralization step, a pH adjustment step is avoided. Another advantage of the invention is that the process time is saved / reduced as degreasing, biolashing and demineralization can be performed simultaneously. Even if combined degreasing, biolashing and demineralization are not carried out as a one-step process, ie at the same time the costs of, e.g. eg acids and human potential for adding acid (s) are saved / reduced as the pH adjustment step between the traditional acid degumming step and the demineralization step is avoided. Compared to simultaneous degreasing and biolash under alkaline conditions, simultaneous degreasing and biolash under acid conditions can remove demineralization over time without further demineralization procedure.

In the context of the invention, the term "treatment" means the combination of enzymes that come from facilitated processing, such as combined degreasing and washing, combined degreasing and biopolishing, combined degreasing and abrasion; etc.

In the context of the invention, the term "biopolishing" is a specific surface treatment of the yarn, which improves fabric quality with respect to handling and appearance without loss of fabric wetting. The most important effects of biopolishing can be characterized by less lint and pellet formation, increased gloss / luster, improved tissue handling, increased durable softness and improved water absorbance.

In the context of the invention, the term "combined" or "combination" means that the combined process steps or combination are performed sequentially or simultaneously in a bath (ie, same treatment solution). In a preferred embodiment, the combined process or combination is performed simultaneously in a bath (i.e. same treatment solution).

In the context of the invention, the term "fabric" is used interchangeably with the term "textile" and means, as opposed to "used" laundry fabric, fabrics, clothing, fibers, yarns or other types of newly manufactured processed, preferably dyed, fabrics. . Fabrics may be constructed of fibers by weaving, weaving or nonwoven operations. Weaving and interlacing require yarn as consumption, while nonwoven fabric is the result of random binding of fibers (paper can be imagined as nonwoven).

The woven cloth is constructed by "padding" by weaving or weft threads between warp threads stretched in the longitudinal direction of the loom.

Warp yarns should be starched prior to weaving in order to lubricate them and protect them from abrasion at the high insertion speed of the filling yarns during weaving. The filler yarn can be woven through the warp yarns in an "over one - under" (single weaving) mode or over "over one" under (twill) or any other myriad permutations. The strength, texture and pattern are related not only to yarn type / quality, but also to weft type. Usually, dresses, shirts, pants, sheets, towels, curtains etc. They are made of nonwoven cloth.

Interlacing is the formation of a fabric by joining together interlocking wire loops. The opposite of weaving, which is constructed of two types of yarn and has many "ends", is produced from a single continuous strand of yarn. As with weaving, there are many different ways to tie the yarn together and the final properties of the fabric are dependent on both yarn and knitting type. Underwear, sweaters, socks, sports shirts, t-shirts etc. are derived from interwoven fabrics.

Nonwoven cloths are fabric sheets produced by bonding and / or interlocking fibers and filaments by mechanical, thermal, chemical or solvent mediated processes. The resulting fabric may be in the form of continuous sheet, laminate or film-like structures. Typical examples are baby diapers, towels, mops, surgical gowns, "environmentally friendly" fashion fibers, filter media, bedding, roofing materials, two-dimensional fabric support and many others. According to the invention, the process may be applied to any starched fabric known in the art (woven, woven or non-woven). The process is applied to newly manufactured starched fabric, as opposed to used and / or dirty fabric to be cleaned during laundry. In one embodiment, the fabric is made of natural and / or man-made fibers. In another embodiment, the fabric is made of animal fibers. In particular, the process of the invention may be applied to cellulose-containing or cellulosic fabrics such as cotton, viscose, rayon, ramie, flax, cellulose acetate, denim, lyocell (Tencel ™, e.g. produced by Courtaulds Fibers) or mixtures thereof, or mixtures of any of these fibers together with synthetic fibers (eg polyester, polyamide, acrylic or polyurethane, nylon, poly (ethylene terephthalate) or poly (lactic acid) or other natural fibers, such as wool and silk, such as viscose and cotton blends, liocel / cotton blends Viscose / wool blends, liocel / wool blends, cotton / wool blends, flax, ramie and other cellulose fiber-based fabrics including all blends of cellulosic fibers with other fibers such as wool, polyamide, acrylic and polyester fibers, eg viscose / cotton / polyester blends, wool / cotton / polyester blends, wool / cotton blends / polyester, acrylic and polyester fibers ter, eg viscose / cotton / polyester blends, wool / cotton / polyester blends, linen / cotton blends etc. The process may also be used in synthetic fabric, e.g. e.g. consisting essentially of 100% polyester, polyamide, nylon respectively. The term "wool" means any commercially useful animal hair product, for example sheep, camel, rabbit, goat, mud wool and known as merino wool, Shetland wool, cashmere wool, alpaca wool, angora goat hair etc. . and includes woolen fiber and animal hair. The process of the invention may be used with wool or animal hair material in the form of staple, fiber, yarn or woven or interwoven.

The alpha amylase used according to the process of the invention may be any acidic alpha amylase, but is preferably of bacterial or fungal origin.

Preferably, the acidic alpha amylase is derived from a filamentous fungus, especially a strain of Aspergillus, Rhizomucor or Meripillus.

The term "acidic alpha amylase" means an alpha amylase (E.C. 3.2.1.1), which has an optimal activity at a pH in the range of 1 to 7, preferably 1 to 5, at a temperature of 50 ° C.

The term "degumming" is to be understood in a conventional manner, that is, the degradation and / or removal of sizing agents such as warp yarns in a woven cloth.

The term "starch-containing fabric or amide derivatives" is intended to indicate any type of fabric, in particular non-woven fabric, prepared from a cellulose-containing material, containing starch or starch derivatives. The fabric is usually dyed and made of cotton, viscose, flax and the like. The major part of the starch or starch derivatives present in the fabric is usually gum with which the threads, usually warp threads, were coated prior to weaving.

The term "carbohydrate binding module (CBM)" or as often referred to as a "carbohydrate binding domain (CBD)" is a polypeptide amino acid sequence which preferentially binds to a poly or oligosaccharide (carbohydrate). ), often - but not necessarily exclusively - to a water-insoluble (including crystalline) form.

Even if not specifically mentioned with respect to the process of the invention, it should be understood that the enzyme (s) or agent (s) are / are used in an "effective amount". The term "effective amount" means an amount of e.g. alpha-amylase which is capable of providing the desired effect, i.e. tissue degumming, as compared to a tissue that has not been treated with said enzyme (s).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to providing a process for degumming a starched fabric during the manufacture of especially new fabrics.

The degumming step of the invention is in a preferred embodiment, followed by a washing step, preferably an enzymatic washing step, preferably with a washing enzyme such as pectinase, e.g. e.g., a pectate lyase, a lipase, a protease, or combination thereof, and a bleaching step, preferably involving hydrogen peroxide bleaching and / or a hydrogen peroxide generating agent. Relevant cleaning processes are described in U.S. Patent No. 5,578,489, U.S. Patent No. 5,912,407 and U.S. Patent No. 6,630,342. Relevant bleaching processes are described in U.S. Patent No. 5,851,233, U.S. Patent No. 5,752,980 and U.S. Patent No. 5,928,380. Combined cleaning and bleaching processes are described in 2003/002810 (Novozymes) and WO 2003/002705 (Novozymes). In accordance with the present invention, the fabric may be degreased and demineralized simultaneously in the same aqueous treatment solution (i.e. a bath) or subsequently in the same or two separate treatment solutions (i.e. one or two baths). In a preferred embodiment, degumming and demineralization are performed simultaneously in the same treatment solution (i.e. a bath). The process of the invention may be carried out using ironing / degreasing equipment, e.g. eg cushion systems, J-boxes, jets, shakers etc. In general, no additional processing equipment is required.

According to the invention, simultaneous degumming and demineralization are performed by incubating starched tissue in an aqueous treatment solution having a pH in the range of 1 to 7, aqueous treatment solution comprising an acidic alpha amylase. In a preferred embodiment, the pH during incubation is in the range 1 to 4, especially between pH 2 and 4.

Woven articles are the predominant form of fabric construction. The weaving process requires a "sizing" of the warp yarn to protect it from abrasion. Unmodified and modified starches, polyvinyl alcohol (PVA), carboxy methyl cellulose (CMC), waxes and acrylic binders and mixtures thereof are examples of typically used sizing agents. The sizing agent may according to the present invention be a starch based or starch derivative based sizing agent, but may also contain one or more non-starch based or starch derived sizing agents. The sizing agent (s) are generally removed after the weaving process as the first step of preparing woven articles.

One or more other agents including stabilizers, surfactants, wetting agents, dispersing agent, sequestering agents and emulsifying agents, or mixtures thereof, may be present during a degumming process of the invention. The starched fabric is allowed to incubate in the aqueous treatment solution for a sufficiently long period of time to perform the degumming of the starched fabric. The optimum period is dependent on the type of processing regime and temperature and may range from about 15 minutes to several days, e.g. eg 48 hours. A process of the invention is preferably carried out at a temperature in the range of 5 to 90 ° C, in particular from 20 to 90 ° C, dependent on the processing regime.

The processing regime can be either batch or continuous, with the fabric being contacted by the open width or rope treatment aqueous stream.

Continuous operations may utilize a saturator whereby an approximate equal width of tissue weight treatment solution is applied to the tissue, followed by a heated residence chamber where the chemical reaction occurs. A wash section then prepares the fabric for the next processing step. In order to ensure high whiteness or good wettability and resulting dyeing, the degumming enzyme (s) and other agents should be completely removed.

Batching processes may occur in a bath (treatment solution) whereby the tissue is contacted with, e.g. approximately 8-15 times its weight of aqueous treatment solution.

After an incubation period, the aqueous treatment solution is drained, the tissue is rinsed and the next processing step is started. Discontinuous PB-processes (ie Buffer Batch processes) involve a saturator, whereby an approximate equal weight of aqueous treatment solution per tissue weight is applied to the tissue, followed by a residence time, which in the case of CPB process (ie Cold Cap Batch process) could be one or more days. For example, a CPB process may be carried out at 20-40 ° C for 8-24 h or more at a pH in the range 1 to 7, preferably at a pH in the range 1 to 4, especially between pH 2 and 4. Further, a PB-process can be performed at 40-90 ° C for 1-6 hours at a pH in the range of about 1 to 7, preferably about 1 to 5, more preferably 1 to 4. , especially between pH 2 and 4.

In one embodiment, the degumming process of the invention may be carried out using an effective amount of alpha-amylase, preferably acidic alpha-amylase and an acid such as acetic acid or sulfuric acid or the like.

Enzymes

Alpha amylases

The alpha amylase (s) used in the process of the invention may be any alpha amylase, preferably of bacterial or fungal origin. In a preferred embodiment the alpha amylase is an acidic alpha amylase or hybrid alpha amylase described in WO 2005/003311, which is hereby incorporated by reference.

In a preferred embodiment, alpha-amylase includes a carbohydrate binding module (CBM) as defined in WO 2005/003311, preferably a 20 CBM family as defined in WO 2005/003311.

Specifically contemplated are CMBs, including those selected from the group consisting of Aspergillus kawachii described in SEQ ID NO: 2; Bacillus flavothermus described in SEQ ID NO: 5: Bacillus sp described in SEQ ID NO: 6; Alcaliphilic Bacillus described in SEQ ID NO: 7; Hormoconis resine described in SEQ ID NO: 8; Lentinula edodes described in SEQ ID NO; 9; Neurospora crassa described in SEQ ID NO; 10; Talaromyces byssochlamydiodes described in SEQ ID NO: 11; Geosmithia cyilndrospora described in SEQ ID NO. 12; Scorias spodiosa described in SEQ ID NO: 13, Eupenicillium ludwigii described in SEQ ID NO; 14; Aspergillus japonicus described in SEQ ID NO. 15; Penicillium cf. miczynskii described in SEQ ID NO: 16; Mz 1 Penicillium sp, described in SEQ ID NO: 17; Thysanospora sp. disclosed in SEQ ID NO: 18; Gray humicola var thermoidea described in SEQ ID NO: 19; Aspergillus niger described in SEQ ID NO; 20; or Althea rolfsii described in SEQ ID NO: 21. Fungal Alpha Amylases

In one embodiment, the fungal alpha-amylase is of yeast or filamentous fungal origin. In a preferred embodiment, the fungal alpha amylase is an acidic alpha amylase.

Preferred alpha-amylases include, for example, alpha-amylases obtainable from Aspergillus species, in particular Aspergillus niger, A. oryzae and A. awamori, A. kawachii, such as the acid alpha amylase described as SWISSPROT P56271, or described in more detail in WO 89/01969 (Example 3). Mature acid alpha amylase has the amino acid sequence shown as 22-511 of SEQ ID NO: 4, encoded by the DNA sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 38. Also preferred are alpha-amylase sequences having more than 50%, such as more than 60%, more than 70%, more than 80% or more than 90%, more than 95%, more than that 96%, more than 97%, more than 98% or even more than 99% identity to the amino acid sequence shown in SEQ ID NOS: 4 or 38, respectively.

In another preferred embodiment, the alpha-amylase sequence is derived from an A. oryzae acid alpha-amylase. More preferably, the alpha-amylase sequence is more than 50%, such as more than 60%, more than 70%, more than 80% or more than 90%, more than 95%, more than that 96%, more than 97%, more than 98%, or even more than 99% identity to the amino acid sequence shown in SEQ ID NO: 4 or 38, respectively.

In another preferred embodiment, the alpha-amylase sequence is derived from an A. oryzae acid alpha-amylase. More preferably, the alpha-amylase sequence is more than 50%, such as more than 60%, more than 70%, more than 80% or more than 90%, more than 95%, more than that 96%, more than 97%, more than 98%, or more than 99% identity to the amino acid sequence shown in SEQ ID NO: 39.

In one embodiment, alpha-amylase is Aspergillus kawachii alpha-amylase described in SEQ ID NO: 37, which in wild-type form contains a carbohydrate binding domain (CBD) also shown in SEQ ID NO: 2 .

In a preferred embodiment, alpha amylase is an alpha amylase having more than 50%, such as more than 60%, more than 70%, more than 80% or more than 90%, more than 95%, more than 96%, more than 97%; more than 98%, or even more than 99% identity to the amino acid sequence shown in SEQ DD NOS: 43, 44, 46 or 47, respectively.

Alpha amylase may be present at a concentration of 1-3,000 AFAU / kg tissue, preferably 10-1,000 AFAU / kg tissue, especially 100-500 AFAU / kg tissue or 1-3,000 AFAU / L treatment solution, preferably 10-1,000 AFAU / L treatment solution, especially 100-500 AFAU / L treatment solution.

Bacterial Alpha Amylases

In one embodiment the alpha amylase is of bacterial origin. In a preferred embodiment the bacterial alpha amylase is an acidic alpha amylase.

Bacterial alpha-amylase is preferably derived from a Bacillus strain such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus sp, such as Bacillus sp. NCIB 12289, NCIB 12512 (WD 95/26397), NCIB 12513 (WO 95/26397), DSM 9375 (WO 95/26397), DSMZ 12648 (WD 00/60060), DSMZ 12649 (WO 00/80060), KSM AP 1378 (WO 97/00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred are Bacillus sp. Alpha-amylases. described in WO 95/26397 as SEQ ID NOS. 1 and 2, respectively, AA560 alpha-amylase described as SEQ ID NO: 2 in WO 00/60060 (ie, SEQ ID NO: 40 here), and Alpha-amylase # 707 described by Tsukamoto et al., In Biochemical and Biophysical Research Communications, 151, pp. 25-31 (1988).

In one embodiment of the invention the bacterial alpha-amylase is alpha-amylase SP722 described as SEQ ID NO: 2 in WO 95/26397 or alpha-amylase AA560 (SEQ ID NO: 40 here). In a preferred embodiment, the precursor alpha-amylase has one or more deletions at or corresponding to the following positions: Dl 83 and Gl84, preferably wherein said alpha-amylase variant further has a substitution at or corresponding to position N195F (using SEQ ID NO: 40 numbering).

In another preferred embodiment, the precursor alpha-amylase has one or more of the following deletions / substitutions or corresponding to the following deletions / substitutions:

Delta (R81-G182); Delta (D183-G184); Delta (D183-G184) + N195F; R181Q + N445Q + K446N; Delta (D183-G184) + R181Q, Delta (Dl83-G184) and one or more of the following substitutions or corresponding to: R118K, N195F, R320K, R458K, especially where the variant has the following mutations: A (D 183 + G184 ) + R11 18K + N196F + R320K + R45 8K (using the numbering of SEQ ID NO: 40).

In another preferred embodiment, alpha-amylase is AA560 alpha-amylase shown in SEQ ID NO: 40, further comprising one or more of the following substitutions M9L, M202L, V214T, M323T, M382Y, E345R or A560 alpha-amylase with all of the following replacements: M9L,

Commercially available alpha-amylase products or products comprising alpha-amylases include product sold under the following trade names: NATALASE ™, STAINZYME ™ (Novozymes A / S), Bioamylase - D (G), BiOAMYLASE ™ L (Bioconndia Ltd.) , KEIMZYM ™ AT 9000 (BiozyM Ges. MbH Austria). PURASTAR ™ ST, PURASTAR ™ HPAmL, PURAFECT ™ OxAm, RAPIDASE ™ TEX (Genencor Int. Inc. USA), KAM (Kao, Japan).

Alpha amylase may be present at a concentration of about 0.05-150 KNU / L treatment solution, preferably 1-100 KNU / L treatment solution, especially 2-20 KNU / L treatment solution or 0.05-150 KNU / Kg of fabric, preferably 1-100 KNU / kg of fabric, especially 2-20 KNU / kg of fabric.

Hybrid Enzyme

The alpha amylase may be in a preferred embodiment an alpha amylase comprising a carbohydrate binding domain (CBD). Such an alpha-amylase with a CBD may be a wild type enzyme (see, e.g., Aspergillus kawachii above) or a hybrid enzyme (fusion protein) as will be further described below. Hybrid enzymes or a genetically modified wild-type enzyme as referred to herein include species comprising an amino acid sequence of an alpha-amylase enzyme (EC 3.2.1.1) linked (i.e. covalently linked) to an amino acid sequence comprising a carbohydrate binding domain (CBD).

CBD-containing hybrid enzymes, as well as detailed descriptions of the preparation and its purification, are known in the art [see, p. WO 90/00609, WO 94/241158 and WO 95/16782, as well as Greenwood et al., Biotechnology and Bioengineering, 1994, 44: 1295-1305]. They can, p. For example, a DNA construct comprising at least one DNA fragment encoding the carbohydrate binding domain linked, with or without a linker, to a DNA sequence encoding the enzyme of interest and culturing it in a host cell may be prepared by transformation into a host cell. the host cell is transformed to express the fused gene.

The resulting recombinant product (hybrid enzyme) - often referred to in the art as a "fusion protein" - can be described by the following general formula:

A-CBD-MR-X

In the latter formula, A-CBD is the N-terminus or C-terminus region of an amino acid sequence comprising at least the carbohydrate binding domain (CBD) alone. MR is the intermediate region (the "linker") and X is the sequence of amino acid residues of a polypeptide encoded by a DNA sequence encoding the enzyme (or other protein) to which the CBD is to be bound.

Component A may be absent (such that A-CBD is a CBD alone, ie it does not comprise amino acid residues other than those constituting CBD) or may be a sequence of one or more amino acid residues (functioning as an extension CBD terminal alone). The linker (MR) may be a bond or a short bond group comprising from about 2 to about 100 carbon atoms, in particular from 2 to 40 carbon atoms. However, MR is preferably a sequence of from about 2 to about 100 amino acid residues, more preferably from 2 to 40 amino acid residues, such as from 2 to 15 amino acid residues.

Component X may constitute the N-terminal or C-terminal region of the total hybrid enzyme.

It will thus be apparent from the above that the CBD of a hybrid enzyme of the type in question may be positioned C-terminally, N-terminally or internally within the hybrid enzyme.

Linker Sequence

The linker sequence can be any suitable linker sequence. In preferred embodiments, the linker sequence is derived from Athelia rolfsii glycoamylase, A. niger glycoamylase, A. kawachii alpha amylase such as a linker sequence selected from the group consisting of the A. niger glycoamylase linker: TGGTTTTATATGSGSVTSTSKTTATASKTSTSTSTS SEQ ID NO: 22), A. kawachii alpha-amylase linker: TTTTTTAAATSTSKAT TSSSSSSAAATTSSS (SEQ ID NO: 23), Athelia rolfsii glycoamylase linker: GATSPGGSSGS (SEQ ID NO: 24), and the PEPT linker: P EPTPEPT (SEQ ID NO: 24) ID NO: 25). In another preferred embodiment, the hybrid enzymes have a linker sequence that differs from the amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 25 by no more. than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than than 3 positions, not more than 2 positions, or even no more than 1 position.

Carbohydrate Binding Domain

A carbohydrate binding domain (CBD), or as often referred to as a carbohydrate binding module (CBM), is a polypeptide amino acid sequence, which preferentially binds to a poly or oligosaccharide (carbohydrate), often - but not necessarily exclusively - to a water-insoluble (including crystalline) form.

CDBs derived from starch degrading enzymes are often referred to as starch binding domains (SBD) or starch binding modules (SBM). SBDs are CDBs that may occur in certain amylolytic enzymes, such as certain glycoamylases or in enzymes such as cyclodextrin glycanotransferases or in alpha amylases. Also, other subclasses of CBDs would encompass, e.g. cellulose binding domains (cellulolytic enzyme CBDs), chitin binding domains (CBDs that typically occur in chitinases), xylan binding domains (CDBs that typically occur in xylanases), mannan binding domains (CDBs) which typically occur in mannanases).

CDBs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially hydrolytic enzymes (hydrolases), which typically comprise a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate binding domain (CDB) to bind to the carbohydrate substrate in question. Such enzymes may comprise more than one catalytic domain and one, two or three CDBs and optionally further comprise one or more polypeptide amino acid sequence regions linking the CBD (s) with the catalytic domain (s) (s), a region of the latter type being indicated as a "linker". Examples of hydrolytic enzymes comprising a CBD - some of which have already been mentioned above - are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases. CDBs have also been found in algae, e.g. for example, in red algae Porphyra purpurea as a nonhydrolytic polysaccharide binding protein.

In proteins / polypeptides where CDBs occur (eg enzymes, typically hydrolytic enzymes) a CBD may be located at the N or C terminus or in an internal position.

That part of a polypeptide or protein (e.g., hydrolytic enzyme) that constitutes a CBD by itself typically consists of more than about 30 and less than about 250 amino acid residues.

The "Family Carbohydrate Binding Module 20" or a CBM-20 module is in the context of the invention defined as a sequence of approximately 100 amino acids having at least 45% homology to the Carbohydrate Binding Module (CBM) of the polypeptide described in Figure 1 by Joergensen et al. (1997) in Biotechnol. Lett. 19: 1027-1031. CBM comprises the last 102 amino acids of the polypeptide, that is, the amino acid subsequence 582 to amino acid 683. The numbering of the Glycoside Hydrolase Families applied in this description follows the design of Coutinho, PM, &. Henrissat B. (1999) CAZy - Carbohydrate-Active Enzymes server at URL; http://afmb.cnrs-mrs.fr/~cazy/CAZY/index.html or alternatively Coutinho, P.M. & Henrissat, B. 1999; The modular structure of cellulases and other carbohydrate-active enzymes; an integrated database approach. In "Genetics, Biochemistry and Ecology of Cellulose Degradation", Κ. Ohmiya, Κ. Hayashi, Κ. Sakka5 Υ. Kobayashi, S. Karita and Τ. Kimura eds., Uni Publishers Co., Tokyo, ρρ. 15 - 23, and Bourne5 Υ, & Henrissat Β. 2001; Glycoside hydrolases and glycosylfransferases; families and functional modules, Current Opinion in Structural Biology 11: 593-600.

Examples of enzymes comprising a CBD suitable for use in the context of the invention are alpha-amylases, maltogenic alpha-amylases, cellulases, xylanases, mananases, arabinofuranosities, acetylesterases and chitinases. Other CBDs of interest in connection with the present invention include CDBs derived from glycoamylases (EC 3.2.1.3) or CGTases (EC 2.4.1.19).

CDBs derived from fungal, bacterial or plant sources will generally be suitable for use in the context of the invention. Preferred are CBDs of fungal origin, more preferably from Aspergillus sp., Bacillus sp., Klebsiella sp. or Rhizopus sp. In this regard, suitable techniques for isolating pertinent genes are well known in the art.

Preferred for the invention are CBDs from the Carbohydrate Binding Module Family 20. CBDs from the Carbohydrate Binding Family 20 suitable for the invention may be derived from Aspergillus awamori glycoamylases (SWISSPROT Q12537), Aspergillus kawachii (SWISSPROT P23176) , Aspergillus niger (SWISSPROT P04064), Aspergillus oryzae (SWISSPROT P36914), from Aspergillus kawachii alpha-amylases (EMBL: # AB008370), Aspergillus nidulans (NCBI AAF17100.1), from Beta-PillusT3636 Bacillus amylases (NCBI AAF17100.1) or Bacillus circulans CGTases (SWISSPROT P43379). Preferred is an Aspergillus kawachii alpha-amylase CBD (EMBL: # AB008370) as well as CBDs having at least 50%, 80%, 70%. 80% or even at least 90%, 95%, 96%, 97%, 98%, or 99% CBD identity of an Aspergillus kawachii alpha-amylase (EMBL: # A8008370), that is, a CBD having at least 50%, 60%, 70%, 80% or even at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 2. Also preferred for the invention are CBDs from the Carbohydrate Binding Module Family 20, having the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 8 and SEQ ID NO: 7 and described in PCT application no. PCT / DK2004 / 000458 (or Danish patent application PA 2003 00949) as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively. Other preferred CBDs include Hormoconis sp glycoamylase CDBs such as Hormoconis resine (syn. Creosoto fungus or Amorphotheca resine), such as SWISSPROT CBD: Q03045: (SEQ ID NO: 8) from Lentinula sp. such as de Lentinula edodes (shiitake mushroom) such as CBD of SPTREMBL: Q9P4C5 (SEQ ID NO: 9), by Neurospora sp. such as from Neurospora crassa such as SWISSPROT: P14804 (SEQ ID NO: 10) CBD from Talaromyces sp, such as from Talaromyces byssochlamyodioides, such as NN005220 (SEQ ID NO: 11) CBD from Geosmithia sp such as of Geosmithia cylindrospora, such as CBD of NN48286 (SEQ ID NO: 12), of Scorias sp, such as Scorias spongiosa, such as CBD of NN007096 (SEQ ID NO: 13), of Eupenicillium sp, such as Eupenicillium ludwigii, such as CBD of NN005968 (SEQ ID NO: 14), from Aspergillus sp. Aspergillus japonicus such as the CBD of NNOO1136 (SEQ ID NO: 15), Peniciliium sp, such as Penicillium cf. miczynskii such as CBD of NN48891 (SEQ ID NO: 16) of Mzl Penicillium sp. such as CBD of NN48690 (SEQ ID NO: 17) from Thysanophora sp. such as the CBD of NN48711 (SEQ ID NO: 18), and Humicola sp. such as Humicola grisea var. thermoidea such as SPTREMBL CBD: Q12623 (SEQ ID NO: 19). Most preferred CBDs include Aspergillus sp. such as Aspergillus niger, such as SEQ ID NO: 20, and Athelia sp. Such as Athelia rolfsii such as SEQ ID NO: 21. Also preferred according to the invention are any CBD having at least 50%, 60%, 70%, 80% or even at least 90%, 95%, 96%. , 97%, 98% or 99% identity to any of the aforementioned CBD amino acid sequences.

In addition, the CDBs of the Carbohydrate Binding Module 20 Family can be found at URL: http: //afmb.cnrsmrs.fr/~cazv/CAZY/index.html.

Once a nucleotide sequence encoding the substrate binding region (carbohydrate binding) has been identified, such as cDNA or chromosomal DNA, it can be engineered in a variety of ways to fuse it to a DNA sequence encoding the enzyme. interest. The DNA fragment encoding the carbohydrate binding amino acid sequence and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA can then be manipulated in a variety of ways for expression.

In one embodiment, the alpha amylase comprised in the hybrid is an alpha amylase described above in the "Alpha Amylase" section. In a preferred embodiment, alpha amylase is of fungal origin. In a more preferred embodiment, alpha amylase is an acidic alpha amylase.

In a preferred embodiment, the carbohydrate binding domain and / or linker sequence is of fungal origin. The carbohydrate binding domain may be derived from an alpha amylase, but may also be derived from a protein, e.g. e.g. enzymes having glycoamylase activity.

In one embodiment the alpha amylase is derived from a strain of Aspergillus, or Athelia. In one embodiment, alpha amylase is derived from a strain of Aspergillus oryzae or Aspergillus niger. In a specific embodiment, alpha-amylase is A. oryzae acid alpha-amylase described in SEQ LD NO: 39. In a specific embodiment, the linker sequence may be derived from an Aspergillus strain, such as such as A. kawachii alpha-amylase (SEQ ID NO: 23) or A. rolfsii glycoamylase (SEQ ID NO: 24). In one embodiment, the CBD is derived from a strain of Aspergillus or Athelia. In a specific embodiment, the CBD is A. kawachii alpha amylase shown in SEQ ID NO: 1 or A. rolfsii glycoamylase shown in SEQ ID NO: 21.

Preferred is the embodiment in which the hybrid enzyme comprises an alpha-amylase sequence derived from the catalytic domain of A. niger acid alpha-amylase, having the sequence shown in SEQ ID NO: 38 and / or a derived linker sequence. A. kawachii alpha amylase shown in SEQ ID NO: 23 or A. rolfsii glycoamylase shown in SEQ ID NO: 24 and / or CBD is derived from A. kawachii alpha amylase shown in SEQ ID NO : 2, from A. rolfsii glycoamylase shown in the sequence SEQ ID NO: 21 or from A. niger glycoamylase shown in SEQ ID NO: 22.

In a preferred embodiment, the hybrid enzyme comprises the catalytic domain of A. niger acid alpha amylase, having the sequence shown in SEQ ID NO: 38, the A. kawachii alpha amylase linker shown in SEQ ID NO. : 23 and the CBD of A. kawachii alpha-amylase shown in SEQ ID NO: 2.

In a specific embodiment, the hybrid enzyme is the mature part of the amino acid sequence shown in SEQ ID NO: 28 (A. kawachii-CBD alpha-amylase alpha-amylase acid catalyst domain niger glycoamylase), SEQ ID NO: 30 (A. kawachii-CBD alpha-amylase acid alpha-amylase catalytic domain of A. rolfsii glycoamylase) or SEQ ID NO: 32 (A. oryzae acid alpha-amylase catalytic domain A. kawachii-CBD alpha-amylase alpha-amylase linker) or SEQ ID NO: 34 (A. acid alpha-amylase catalytic domain A. rolfsii-CBD glycoamylase niger-ligand-glyceramylase A. rolfsii-CBD niger-linker or SEQ ID NO: 36 (A. oryzae acid alpha-amylase catalytic domain of A. rolfsii- CBD-A glycoamylase glycosylase linker rolfsii) or the hybrid consisting of the catalytic domain of A. niger acid alpha-amylase (SEQ ED NO: 4 or 38, respectively) -glyca linker A. kawachii milase (SEQ ID NO: 23) - A. kawachii glycoamylase CBD (SEQ ID NO: 2) or a hybrid enzyme having an amino acid sequence having at least 50%, 60%, 70%, 80% or even at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the above amino acid sequences.

In another preferred embodiment the hybrid enzyme has an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 28 (A. niger acid alpha-amylase alpha-amylase linker catalytic domain. A. niger glycoamylase kawachii-CBD), SEQ ID NO: 30 (A. niger acid alpha-amylase catalytic domain A. kawachii- A. rolfsii glycoamylase alpha-amylase linker), SEQ ID NO: 32 (A. oryza acid alpha-amylase catalytic domain- A. kawachii-CBD alpha-amylase alpha-amylase linker), SEQ ID NO: 34 (acidic alpha-amylase catalytic domain niger-CBD glycosamylase assay from A. rolfsii) or SEQ ID NO: 36 (A. oryzae acid alpha-amylase catalytic domain - A. niger acid alpha amylase catalytic domain (SEQ ID NOS: 4 or 38, respectively) - A. kawachii glycoamylase linker (SEQ ID NO: 23) - A. kawachii glycoamylase linker (SEQ ID NO: 2) at no more than 10 positions No more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions , not more than 2 positions, or even no more than 1 position.

Preferably, the hybrid enzyme comprises a CBD sequence having at least 50%, 60%, 70%, 80% or even at least 90%, 95%, 96%, 97%, 98%, or 99% identity with any one. one of the amino acid sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO : 20 or SEQ ED NO: 21. Even more preferred the hybrid enzyme comprises a CBD sequence having an amino acid sequence shown in SEQ ED NO: 5, SEQ ED NO: 8, SEQ ED NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, in yet another preferred embodiment, the CBD sequence has an amino acid sequence that differs from of the sequence of am acidic acid shown in SEQ ED NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 at no more than 10 amino acid positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 8 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.

In a most preferred embodiment, the hybrid enzyme comprises a CBD derivative of an A. rolfsii glycoamylase, such as A. rolfsii AHU 9627 glycoamylase described in U.S. Patent No. 4,727,026.

Acid Washing Enzymes

Any acid washing enzyme may be used in accordance with the present invention. The acid wash enzyme may be an acid enzyme selected from the group consisting of pectinase, cellulase, lipase, protease, xyloglycanase, cutinase and a mixture thereof. A washing enzyme is "acidic" in the context of the present invention when the optimum pH under the conditions present during degreasing and washing simultaneously is below 7, such as between 1-7, preferably below 5, such as between 1 - 5, especially below 4, such as between 1-4.

Several washing enzymes are known as:

Polygalacturonase (EC 3.2.1.15) catalyzes the random hydrolysis of 1,4-alpha-D-galactosiduronic bonds in pectate and other galacturonans. Examples of other names are: Pectin Depolymerase; pectinase; endopolygalacturonase; endo polygalacturonase; and endogalacturonase. The systematic name is pol (1,4-alpha-D-galacturonide) glycanhydrolase.

Pectin lyase (EC 4.2.2.10) catalyzes the eliminative cleavage of (1,4) -alpha-D-galacturonan methyl ester to provide oligosaccharides with 4-deoxy-6-0-methyl-alpha-D-galact-4 groups enuronisil at its non-reducing ends. Examples of other names are: Pectin transeliminase; polymethylgalacturonic transeliminase; and pectin methyltranseliminase. The systemic name is (1,4) -6-0-methyl-alpha-D-galacturonane lyase.

Pectate lyase (EC 4.2.2.2) catalyzes the eliminative cleavage of (1,4) -alpha-D-galacturonane to provide oligosaccharides with 4-deoxy-alpha-D-enuronosyl groups at their non-reducing ends. Examples of other names are: pectate transeliminase; polygalacturonic transeliminase; and endopectin methyltranseliminase. The systematic name is (1,4) -alpha-D-galacturonane lyase.

Pectinesterase (EC 3.1.1.11) catalyzes the reaction: pectin + η H2O = η methanol + pectate. Examples of other names are: Pectin Demethoxylase; pectin methylesterase; and pectin methyl esterase. The systematic name is pectin pectylhydrolase.

Pectate dissaccharide lyase (EC 4.2.2.9) catalyzes the eliminative cleavage of 4- (4-deoxy-alpha-D-galact-4-enuronosyl) -D-galacturonate from the reducing end of the pectate, i.e., deesterified pectin. Examples of other names are: Pectate exo-lyase; exopectic acid transeliminase; exopectate lyase; and exopolygalactoronic acid trans-eliminase. The systemic name is (1-4) alpha-D-galacturonan reducing end disaccharide lyase.

EC numbering is in accordance with the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzyme-Catalyzed Reactions, published in Enzyme Nomenclature 1992 (Academic Press, San Diego, California, with Supplement 1 (1993), Supplement 2 (1994), Supplement 3 (1995), Supplement 4 (1997) and Supplement 5 (in Eur. J. Biochem. 1994, 223: 1-5; Eur. J. Biochem. 1995, 232: 1-6; Eur. J. Biochem. 1996, 237: 1-5: Eur. J. Biochem. 1997, 250: 1-6 and Eur. J. Biochem. 1999, 264: 610-650: respectively).

In a preferred embodiment, the acid pectinase is a pectate lyase, a pectin lyase, a polygalactoronase or a pligalactoronate lyase.

The term "pectinase" includes any acid pectinase enzyme. Pectinases are a group of enzymes that hydrolyze glycosidic bonds of pectic substances, mainly poly-1,4-alpha-D-galacturonide and their derivatives (see reference Sakai et al., Pectin, pectinase and propectinase: production, properties and applications, in: Advances in Applied Microbiology, Vol. 39, pp. 213-294 (1993)) whose enzyme is understood to include a mature protein or precursor form or functional fragment thereof, which essentially has the activity of the full-length enzyme. In addition, the expression pectinase enzyme is intended to include homologues or analogs of such enzymes.

Preferably, acid pectinase is an enzyme that catalyzes the random cleavage of alpha-1,4-glycosidic bonds in pectic acid, also called polygalacturonic acid, by transelimination, such as enzyme class polygalacturonate lyase (EC 4.2.2.2) (PGL ), also known as poly (1,4-alpha-D-galacturonide) lyase, also known as pectate lyase. Also preferred is a pectinase enzyme, which catalyzes the random hydrolysis of alpha-1,4-glycosidic bonds in pectic acid, such as enzyme class polygalactoronase (EC 3.2.1.15) (PG), also known as endo-PG. Also preferred is a pectinase enzyme such as polymethylgalacturonate lyase (EC 4.2.2.10) (PMGL), also known as Endo-PMGL, also known as poly (methoxygalactoronide) lyase, also known as pectin lyase, which catalyzes the random cleavage of alpha-1,4-glycosidic linkages of pectin.

Other preferred pectinases are galactanases (EC 3.2.1.89), arabinanases (EC 3.2.1.99), pectin esterases (EC 3.1.1.11) and mannanases (EC 3.2.1.78).

For purposes of the invention, the source of the above enzymes including pectin lyase, pectate lyase and pectinesterase is not critical, e.g. e.g., enzymes may be obtained from a plant, an animal or a microorganism such as a bacterium or fungus, e.g. eg, a filamentous fungus or yeast. Enzymes may e.g. be obtained from these sources by the use of recombinant DNA techniques as is known in the art. The enzymes may be natural or wild type enzymes or any mutant, variant or fragment thereof exhibiting pertinent enzymatic activity as well as synthetic enzymes such as scrambled enzymes and consensus enzymes. Such genetically engineered enzymes may be prepared as is generally known in the art, e.g. by site-directed mutagenesis, by PCR (using a PCR fragment containing the desired mutation as one of the primers of the PCR reactions), or by Random Mutagenesis. The preparation of consensus proteins is described, e.g. e.g. in EP 897985.

Pectinase may be a component occurring in an enzyme system produced by a given microorganism, such as an enzyme system primarily comprising several different pectinase components, including those identified above.

Alternatively, pectinase may be a single component, that is, a component essentially free of other pectinase enzymes, which may occur in an enzyme system produced by a given microorganism, the only component typically being a recombinant component, that is, produced by cloning a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host. Such useful recombinant enzymes, especially pectinase, pectin lyases and polygalacturonases are described in detail in, e.g., WO 93/020193, WO 02/092741, WO 03/095638 and WO 2004/092479 (from Novozymes A / S), which are hereby incorporated by reference in their entirety, including sequence listings. The host is preferably a heterologous host, but the host may, under certain conditions, also be the homologous host.

In a preferred embodiment, the pectinase used according to the present invention is derived from the genus Aspergillus.

In a still preferred embodiment, pectinase is protopectinase having an amino acid sequence SEQ ID NO: 1 of JP 11681877 or protopectinase having an amino acid sequence generated by deletion, substitution or insertion of one amino acid or several amino acids. acids of the amino acid sequence and having an activity at or above the level of protopectinase activity with the amino acid sequence of SEQ ID NO: 1 of JP 11682877.

Pectinase, such as especially pectate lyase, may preferably be present in a concentration in the range of 1 - 1500 APSU / kg of tissue, preferably 10 - 1200 APSU / kg of tissue, especially 100 - 1000 APSU / kg of tissue. Commercially available acidic pectate lyases according to the present invention include Pectinex® BE XXL, Pectinex® BE Color, Pectinex® ultra; Pectinex ™ Ultra SP-L5 Pectinex® Yield Mash, Pectinex® XXLm Pectinex® Smash XXL, Pectinex® Smash, Pectinex ™ AR from Novozymes A / S, Denmark.

Proteases

Any protease suitable for use in acidic solutions may be used. Suitable proteases include those of animal, plant or microbial origin. Microbial origin is preferred. Chemically or generically modified mutants are included. The protease may be a serine protease, preferably an acid microbial protease or a trypsin-like protease. Examples of acid proteases are subtilisins, especially those derived from Bacillus, preferably Bacillus lentus or Bacillus clausii, e.g. subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).

Preferred commercially available protease enzymes include those sold under the trade names ALCALASE ™, SAVINASE ™ 16 L Type Ex, PRIMASE ™, DURAZYM ™, and ESPERASE ™ (Novozymes A / S, Denmark), those sold under the trademark OPTICLEAM ™, OPTIMASE ™, PROPARASE ™, PURAFECT ™, PURAPECT ™ MA and PURAPECT ™ OX, PURAFECT ™ OX-I and PURAFECT ™ OX-2 by Genencor International Inc., (USA).

In one embodiment of the process of the invention, the protease may be present at a concentration of 0.001-10 KNPU / L, preferably 0 1-1 KNPU / L. especially around 0.3 KNPU / L or 0.001-10 KNPU / kg fabric, preferably 0.1-1 KNPU / kg fabric, especially around 0.3 KNPU / kg fabric.

Lipases

Any lipase suitable for use in acidic solutions may be used. Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.

Examples of useful lipases include representative acid lipase enzymes including Lipolase.TM., Lipolase.TM. Uitra, Palatase.TM. A, Palatase.TM. M and Lipozima.TM, commercially available from Novo industri A / S. These acid lipase enzymes are 1,3-specific lipase enzymes which hydrolyze fatty acid at the 1 and 3 positions of the triglyceride. Another representative acid lipase enzyme is BCC Yeast Lipase commercially available from Bio-Cat, Inc. This enzyme is derived from a selected strain of Candida cylindracea and is a nonspecific lipase enzyme that hydrolyzes fatty acid in all three triglyceride positions.

In one embodiment of the process of the invention, a lipase enzyme may be present in a concentration of 0.01 - 100 LU / L of treatment solution, preferably 1-10 LU / L of treatment solution, especially around 1 LU / L treatment solution or 0.01-100 LU / kg fabric, preferably 1-10 LU / kg fabric, especially around 1 LU / kg fabric. Cellulases

In the present context, the term "cellulase or" cellulolytic enzyme "refers to an enzyme that catalyzes the degradation of cellulose into glucose, cellobiose, triose and other celloligosaccharides. Cellulose is a beta-1 bonded glucose polymer Cellulose chains form numerous intra and intermolecular hydrogen bonds, which result in the formation of insoluble cellulose microfibrils. Microbial hydrolysis of glucose to cellulose involves the following three major classes of cellulases: endo-1,4-beta -glycanases (EC 3.2.1.4), which cleave beta-1,4-glycosidic bonds randomly across all cellulose molecules, cellobiohydrolases (EC 3.2.1.91) (exoglycans), which digest non-reducing end cellulose, and beta-glycosidases (EC 3.2.1.21), which hydrolyze cellobiose and low molecular weight cellodextrins to release glucose Most cellulases consist of a cellulose binding domain (CBD) and a catalyst (CD) separated by a proline-rich linker and hydroxy amino acid residues. In the report and claims, the term "endoglycanase" is intended to indicate enzymes with cellulolytic activity, especially endo-1,4-beta-glycanase activity, which are classified in EC 3.2.1.4 according to Enzyme Nomenclature (1992) and are capable of catalyzing (endo) hydrolysis of 1,4-beta-D-glycosidic linkages in cereal cellulose, lichenine and beta-D-glycans, including 1,4-beta-D-glycan linkages, also containing 1-bonds, 3 Any cellulase suitable for use in acidic solutions may be used. Suitable cellulases include those of bacterial or fungal origin. Chemically or generically modified mutants are included. Suitable cellulases are described in U.S. Patent No. 4,435,307, which describes fungal cellulases produced from Humicola insolens. Especially suitable cellulases are cellulases having color care benefits. Examples of such cellulases are the cellulases described in European patent application No. 0 495 257, WO 91/17243 and WO 96/29397.

The specific acid cellulase enzyme for hydrolysis of polymeric cellulose produced by Acetobacter bacteria may be derived from certain strains of Trichoderma reesei or Aspergillus niger, or their naturally or artificially induced mutants or variants. As used herein, Trichoderma reesei indicates known microorganisms by that name, as well as those microorganisms classified under the names Trichoderma longibrachiatum and Triochoderma viride. Any cellulase enzyme or enzyme complex that is specific for hydrolysis of cellulose produced by Acetobacter bacteria may be used.

A representative acid cellulase enzyme is the Cellulase Tr Concentrate and Multi-enzyme Acid Cellulase complex, which is commercially available from Solvay Enzymes, Inc. Cellulase Tr Concentrate is a food grade cellulase complex obtained by controlled fermentation of a selected strain of Trichoderma reesei. This enzyme complex consists of both exoglycans and endoglycans that directly attack native cellulose, native cellulose derivatives and soluble cellulose derivatives. This enzyme complex specifically hydrolyzes the beta-D.4-glycosidic bonds of bacterial cellulose, in particular the polymeric bacterial cellulose produced by the bacterium Acetobacter, as well as its oligomers and derivatives (U.S. Patent No. 5,975,095).

Another representative cellulase enzyme commercially available from Solvay enzymes, Inc. is the Cellulase TR1 complex and multi-enzyme liquid cellulase. The TRL cellulase cellulose enzyme complex is derived from Trichoderma reesei in the same way as the Cellulase Tr Concentrated enzyme complex, but is prepared and sold in liquid form. Its activity against bacterial cellulose has been shown to be equivalent to that of the enzyme complex and Cellulase Tr Concentrate.

Other enzymes suitable for use in the present invention include Celluzyme Acid P and Celluclast 1.5 L enzyme, both commercially available from Novo Nordisk; Multifect TM. Cellulase 300 enzyme, commercially available from Genencor International and Rapidase RTM. Acid Cellulase enzyme, commercially available from Gist-Brocades Β. V. Still other cellulase enzymes or cellulase enzyme complexes are suitable for use in the present invention as long as they exhibit specific hydrolytic activity directed to the beta-glycosidic bond characteristic of polymeric bacterial cellulose produced by microorganisms such as Acetobacter bacteria (US Patent No. 5,975,095).

In one embodiment of the process of the invention, the cellulase may be used at a concentration in the range of 0.001-10 g of enzyme protein / 1 treatment solution, preferably 0.005-5 g of enzyme protein / 1 treatment solution, especially 0.01-3 g enzyme protein / 1 solution or 0.001-10 g enzyme protein / kg tissue, preferably 0.005-5 g enzyme protein / kg tissue, especially 0.01-3 g enzyme protein / kg tissue. In one embodiment the cellulose is used at a concentration of 0.1-1,000 ECU / g tissue, preferably 0.5-200 ECU / g tissue, especially 1-500 ECU / g tissue. Cutinase

A cutinase is an enzyme capable of degrading the cutin, cf., e.g. Lin T S & Kolattukudy P E, J. Bacteriol., 1978, 133 (2): 942-951. Cutinases, for example, differ from classical lipases in that no measurable activation around the critical micelle concentration (CMC) of the tributyrin substrate is observed. In addition, cutinases are considered to belong to a class of serine esterases. Cutinase may also be a Humicola insolens-derived cutinase described in WO 96/13580. Cutinase may be a variant such as one of the variants described in WO 00/34450 and WO 01/92502, which are hereby incorporated by reference.

Examples of cutinases are those derived from Humicola insolens (U.S. Patent No. 5,827,719); from a strain of Fusarium, e.g. F. roseum culmorum or particularly F. solani pisi (WO 90/09446; WO 94/14964, WO 94/03578). Cutinase may also be derived from a strain of Rhizoctonia, e.g. e., R. solani, or an Alternaria strain, e.g. A. brassicicola (WO 94/03578) or variants thereof, such as those described in WO 00/34450 or WO 01/92502. The cutinase may also be of bacterial origin, such as a strain of Pseudomonas, preferably Pseudomonas mendocin described in WO 01/34899.

Cutinase may be added at a concentration of 0.001 - 25,000 micrograms enzyme protein / gram tissue, preferably 0.01-10,000 micrograms enzyme protein / g tissue, especially 0.05-1000 micrograms enzyme protein / g tissue. Xyloglycanase

Xyloglycanase is a specific xyloglycan enzyme capable of catalyzing xyloglycan solubilization in xyloglycan oligosaccharides. According to IUBMB Enzyme Nomenclature (2003) a xyloglycanase is classified as EC 3.1.1.151. Pauly et al, (Glycobiology, 1999, 9:93 -100) describe an Aspergillus aculeatus xyloglycan-specific endo-beta-1,4-glycanase. A xyloglycanase used according to the present invention may be derived from microorganisms such as fungi or bacteria. Examples of useful xyloglycanases are hydrolysing endoglycans of family 12 xyloglycan, in particular hydrolysing endoglycans of family 12 xyloglycan obtained from, e.g. Aspergillus aculeatus as described in WO 94/14953. Another useful example is a xyloglycanase produced by Trichoderma, especially EGIII. Xyloglycanase may also be derived from a bacterium of the genus Bacillus, including Bacillus licheniformis, Bacillus agaradharens or Bacillus firmus. Xyloglycanase may also be an endoglycanase with xyloglycanase activity and low activity relative to insoluble cellulose and high activity relative to soluble cellulose, e.g. family 7 endoglycanases obtained from e.g. eg Humicola insolens.

The xyloglycanase may be added at a concentration of 0.001 - 25,000 μg enzyme protein / gram tissue, preferably 0.01-10,000 microgram enzyme protein / g tissue, more preferably 0.05-1000 microgram enzyme protein / g tissue, in particular 0.5-500 micrograms enzymatic protein / gram tissue. Composition of the Invention

In the second aspect the invention relates to a composition suitable for use in the process of the invention. The composition may be a solid or liquid (aqueous) composition and may be a concentrated composition or a ready-to-use composition.

Thus, in this aspect, the invention relates to a composition comprising an acidic alpha amylase and an acid scavenging enzyme.

The enzymes comprised may preferably be those mentioned in the "Enzymes" section above.

In one embodiment, the acid alpha amylase is derived from a strain of Bacillus sp, preferably a strain of B. licheniformis, B. amyloliquefaciens, B. stearothermophilus, Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, or DSMZ no. 12849, KSM AP1378, or KSM K36 or KSM K38.

Bacillus alpha-amylase may be a variant having one or more deletions at positions D183 and Gl 84, respectively, and may further have a substitution at position N195F (using the numbering of SEQ ID NO: 4). The Bacillus alpha-amylase variant may also be one having one or more deletions at position D1 83 and G184 and may further have one or more of the following substitutions: R118K, N195F, R320K, R458K (using SEQ ID NO numbering : 6).

Washing enzymes are selected from the group consisting of: pectinase, cellulase, lipase, protease, cutinase, acid xyloglycanase and mixtures thereof.

In a preferred embodiment, the acid pectinase is a peccate lyase, preferably a pectate lyase derived from a Bacillus strain, preferably a B. licheniformis, B. alcalophilus, Bacillus pseudoalcalophilus and Bacillus clarikia strain, especially the Bacillus licheniformis species. Other suitable agents for the process to be carried out may be added separately or included in the composition of the invention. Examples of such agents include stabilizing, surfactant, wetting, dispersing, sequestering and emulsifying agents and mixtures thereof.

Although acid alpha amylase and acid wash enzyme may be added as such, it is preferred that it be formulated in a suitable composition. Thus, the enzymes may be used in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry or in a protected form. Dust-free granules can be produced, e.g. as described in U.S. Patent Nos. No. 4,106,991 and 4,661,452 (both from Novozimes A / S) and may optionally be coated by methods known in the art.

Liquid enzyme preparations may, for example, be stabilized by adding a polyol, such as e.g. propylene glycol, a sugar or sugar alcohol or acetic acid according to established methods. Other enzyme stabilizers are well known in the art. Protected enzymes may be prepared according to the method described in EP 238 216.

In principle, the composition of the invention comprising an acidic alpha-amylase and a washing enzyme may contain any other agent to be used in the combined process of the invention.

The composition of the invention comprises in a preferred embodiment at least one other component selected from the group consisting of stabilizers, surfactants, wetting agents, dispersing agents, sequestering agents and emulsifying agents. All such other components suitable for textile use are well known in the art.

Suitable surfactants include those mentioned in the "Detergent" section above. The wetting agent serves to improve the wettability of the fiber whereby rapid and uniform degreasing and washing can be obtained. The emulsifying agent serves to emulsify hydrophobic impurities present in the fabric. The dispersing agent is to prevent extracted impurities from redepositing in the fabric. The sequestering agent serves to remove ions such as Ca, Mg and Fe, which may have a negative impact on the process and preferred examples include caustic soda (sodium hydroxide) and soda ash (sodium carbonate).

Use of the Invention Composition

In the third aspect the invention relates to the use of the composition of the invention in a simultaneous degreasing and washing process, preferably the process of the invention. In a preferred embodiment, the composition of the invention is used in a process of the invention.

The invention described and claimed herein is not intended to limit the scope by the specific embodiments described herein, as these embodiments are illustrations of various aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, this description including definitions will govern.

Various references are cited herein, the descriptions of which are incorporated by reference in their entirety.

Materials & Methods

Enzymes

Acid Amylase A: Wild type acid alpha amylase derived from A. niger described in SEQ ID NO: 38.

Acid Amylase B: Hybrid alpha-amylase shown in SEQ ID NO: 48, comprising a Rhizomucor pusillus alpha-amylase catalytic domain (CD) having an A. niger carbohydrate binding domain (CBD). - Acid Pectinase A (Pectinex BEE XXL, Novozymes (A / S)): a pectolytic liquid enzyme preparation produced by Aspergillus species.

- Acid Pectinase B (Pectinex Ultra; Novozymes A / S): A highly active pectolytic enzyme preparation containing a range of hemicellulolytic activities produced by a selected strain of Aspergillus aculeatus.

Enzyme classification numbers (EC numbers) referred to in this specification with claims are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc, 1992.

Fabric

- Acid Pectinase C (Pectinex Yield Mash, Novozymes A / S)

- Acid Pectinase D (Pectinex XXL, Novozymes A / S)

- Acid Pectinase E (Pectinex Smash XXL, Novozymes A / S).

- 460U Interlock Knits (Testfabrics, Inc.)

- Vlisco fabric (by Vlisco Helmond Β. V.)

Plug

Citrate Buffer

1) 10 mM Citrate buffer (pH 3.0)

1.954 g citric acid monohydrate and 0.206 g citric acid dihydrate

Sodium citrate are dissolved in 1 L of deionized water.

2) 10 mM Citrate Buffer (pH 4.0)

1.376 g citric acid monohydrate 1.015 g hydrochloric acid dihydrate

Sodium citrate are dissolved in 1 L of deionized water.

Methods:

Determination of Homology

For purposes of the present invention, the degree of homology is determined as the degree of identity between two amino acid sequences as determined by the Clustal method (Higgins, 1989, CABIOS 5: 151 - 153) using LASERGENE ™ MEGALiGN ™ software. (DNASTAR, Inc., Madison, WI) with an identity table and the following multiple alignment parameters: Span Loss 10 and Span Length Loss 10. The double alignment parameters were Ktuple = I, Span Loss = 3, windows = 5, and diago-nals = 5.

Acid Alpha Amylase Activity (AFAU Assay)

When used in accordance with the present invention, the activity of any acidic alpha-amylase may be measured in AFAU (Acid Fungal Alpha-Amylase Units), which are determined relative to an enzyme standard. 1 AFAU is defined as the amount of enzyme that degrades 5260 mg of starch dry matter per hour under the standard conditions mentioned below.

Acid alpha-amylase, an endo-alpha-amylase (1,4-alpha-D-glycan glycan hydrolase, EC 3.2.1.1) hydrolyzes alpha-1,4-glycosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths. The intensity of the color formed with iodine is directly proportional to the starch concentration. Amylase activity is determined using inverse colorimetry as a reduction in starch concentration under the specified analytical conditions.

ALPHA-AMYLASE

<formula> formula see original document page 42 </formula>

Standard Conditions / Reaction Conditions:

Substrate: Soluble starch, ap. 0.17 g / l

Buffer: Citrate, approx. 0.03M

Iodine (12): 0.03 g / l

CaCl2: 1.85 mM

pH: 2.50 ± 0.05 Incubation Temperature: 40 ° C

Reaction Time: 23 seconds

Wavelength: 590 nm

Enzyme concentration: 0.025 AFAU / ml

Enzyme work range: 0.03 - 0.04 AFAU / ml

A leaflet EB-SM-0259.02 / 01 describing this analytical method in more detail is available on request from Novozimas A / S, Denmark, which leaflet is hereby included by reference.

Alpha Amylase (FAU) Activity

Alpha amylase activity can be determined using (4,6-ethylidene (G7) -p-nitrophenyl (G 1) -a, D-maltoeptaoside (ethylidene-G7PNP) as substrate. This method is based on the decomposition of ethylidene-G7PNP by the enzyme in glucose and yellow-colored p-nitrophenol The rate of p-nitrophenol formation can be observed by Konelab 30. This is an expression of the reaction rate and thus enzymatic activity.

Enzyme activity is determined relative to an enzyme standard. 1 FAU is defined as the amount of enzyme that degrades 5,260 mg of starch dry matter per hour under the standard conditions mentioned below.

<table> table see original document page 43 </column> </row> <table>

Leaflet EB-SM-0216.02 describing this analytical method in more detail is available on request from Novozimes A / S, Denmark, which leaflet is hereby included by reference. Determination of PECTIN TRANSELIMINASE (UPTE) ACTIVITY The activity of acid pectinase can be determined by degrading an Obipectin solution to an enzyme standard under the conditions given below: 0.5% Obipectin Reaction:

Substrate concentration: 30 ° C

Temperature: 30 ° C

pH: 5.4

Reaction time: 10 minutes Absorbance: 238 nm

A unit of pectin transeliminase (UPTE) is defined as the amount of enzyme that increases absorbance by 0.01 unit of absorbance per minute under standard conditions.

A leaflet EB-SM-0368.02 / 01 describing this analytical method in more detail is available on request from Novozimes A / S, Denmark, which leaflet is hereby included by reference. Determination of Polygalacturonase (PGU) Activity

<table> table see original document page 44 </column> </row> <table>

Under degradation of polygalacturonic acid, viscosity will be reduced, which is proportional to the polygalacturonase activity of unknown samples.

Leaflet EB-SM-0615.02 describing this analytical method in more detail is available on request from Novozimes A / S, Denmark, which leaflet is hereby included by reference. Degreasing (Tegewa Method)

Starch gum residue is determined visually by comparing an iodine dyed fabric sample strip to a standard set of 1 - 9 scale photos, where 1 is dark blue and 9 is colorless. The colored iodine solution is made by dissolving 10 g KI in 10 ml water, adding 0.635 g I2 and 200 ml ethanol in deionized water to produce a total solution of 11. A tissue sample is cut and immersed in iodine solution for 60 seconds and rinsed in deionized water for about 5 seconds. The tissue sample is classified by at least two professionals after excess water of the sample is compressed out. An average number is provided. The method and standard scales obtainable from Verband TEGEWA, Karistrasse 21, Frankfurt a.M., Germany.

Pectin Removal

The tissue pectin residue was quantitatively determined. The principle is that ruthenium red binds to polyanionic compounds such as unmethylated pectin. The pectin level in the tissue is proportional to the ruthenium red concentration in the cotton tissue, which is linearly proportional to the Kulbelka-Munk function (ie, K / S). The color reflectance (R) of ruthenium dyed fabric was measured at 540 irai (Macbeth colorimeter, Model # CE-7000) and automatically calculated to a K / S value by:

K / S = (1-R) 2 / 2R).

% Pectin removal was calculated using the following formula:

% -removal pectin = 1-% Pectin Res = 1-100 * (K / S-K / S0) / (K / S 100-K / s0

wherein K / Sioo was 100% pectin tissue, typically original untreated tissue, while K / S0 was tissue with 0% residual pectin, typically heavily washed and bleached tissue. Based on information from John H. Lufit and described in a 1971 article "Ruthenium red and Violet I. Chemistry", the dyeing solution was prepared by dissolving 0.2 g / l ruthenium red, 10, g / l of ammonium chloride, 2.5 ml / l of 28% ammonium hydroxide solution, 1.0 g / l of Silwet L-77 and 1.0 g / l of Tergitol 15-S-12 in distilled water, to produce a total solution of 1 liter. The solution was produced daily before use. During dyeing, 100 ml of dye solution was used for 1 gram of tissue. The tissue sample strips were incubated in red ruthenium solution for 15 minutes at room temperature. The sample strip was rinsed on a stretcher and then rinsed in distilled water (100 ml / l g tissue) at 60 ° C for 10 minutes. Color reflectance was measured after drying.

Tissue wettability

Tissue wettability was measured using a drop test method according to the AATCC 79-1995 test method. A drop of water was allowed to fall from a fixed height (1 cm) onto the strained surface of a test specimen. The time required for specular reflection of the water drop to disappear was measured and recorded as wetting time.

Absorption test

The absorption height of textiles is one of the indicators for absorbance. Cut a 25 cm rectangular strip of fabric sample (warp and weft direction) χ 4 cm. If the sample is not available in this size for testing, adjust the method to fit the sample. Using a waterproof / dye-proof pen, draw a line across the top of the sample 1.5 cm from the top of the sample strip and 3 cm from the bottom of the sample. Draw a line through the sample 19 cm from the base of the sample strip. Attach a paper clip with a weight to the base of the fabric. Place the top of the sample strip in the center of the thermometer clamp so that the line is at the base of the clamp. Fill a beaker about half (at least 5 cm above the base of the beaker) with 1 g / l dye solution (eg reactive blue). Adjust the clamp with the sample strip until the surface of the dye solution is flush with the line at the base of the fabric. Start the timer as soon as the sample strip is in position. Measure the height when the dye solution has been completely absorbed from the surface of the dye solution after 30 min. Remove the sample strip and allow it to air dry on a flat surface.

EXAMPLES

Example 1

Washing cotton fabric with acid pectinase A

A 100% 460U cotton fabric was purchased from Test Fabrics. The tissue sample strips were cut to about 2 g each.

Two tampons were produced for this study. Buffer pH 3 was produced by dissolving 1.954 g citric acid monohydrate and 0.206 g sodium citrate dehydrate in 1 liter deionized. The pH 4 buffer was produced by dissolving 1.376 g citric acid monohydrate and 1.015 g sodium citrate dehydrate in 1 liter deionized. Washing was conducted with a Lab-O-Mat. The beaker was filled with 40 ml buffer and two pieces of pre-cut tissue.

1. Pre-rinse: The wetting agent, Leophan, was added to the buffer at a concentration of 0.25 g / l. Then the temperature was raised to 40 ° C for pre-rinsing. After 10 min, the liquid was drained.

2. Biolava: The pre-rinsed beaker was filled with 40 ml of buffer. Acid pectinase was added to each beaker as specified. In the meantime, the second wetting agent, Keirlon Jet B, was dosed at a concentration of 1 g / l. The temperature was raised to 55 ° C and held for 30 min.

3. Inactivation: After the required time reached, add the Dekol NS from the machine / beaker then raise the temperature to 95 ° C and perform for 15 min, lower the temperature to 10 ° C, drained.

4. Hot rinse: Filled with water and incubated at 70 ° C for 10 min.

5. Cold rinse: Filled with cold water and rinsed for 10 min.

6. Water withdrawal by spinning the fabrics and air drying.

7. Measured residual pectin and wetting time of treated tissues.

The test result is shown in Table 1.

Example 2

Wash Cotton Fabric with Acid B Pectinase

The same tissue sample strip and buffers were prepared as in Example 1. Acid Pectinase B had different enzyme composition compared to Acid Pectinase A. The pectin removal performance was shown in Table 1. Both enzymes performed well. at acidic pH's.

Table 1

<table> table see original document page 48 </column> </row> <table>

Example 3

Simultaneous degreasing and bio-washing of Cold Cap Batch with

Acid A Amylase and Acid A Pectinase

The Vilisco fabric (100% cotton) was from Vilisco and cut to 5 cm * 15 cm. Buffer pH 3 and pH 4 were prepared following the procedures described in Example 1. 10 ml buffer was added to a beaker, Keirlon Jet B was added at a concentration of 2 g / l. Enzymes (doses were listed in Table 2) were added to the impregnation solution and mixed well. 2 attached sample strips of the same tissue on a pair of forceps. Immerse the sample strips in the soaking bath for 30 seconds and fill it with the pader (Mathis Inc. U.S.A.). Repeated soaking and squeezing for a longer time to ensure 100% moisture absorption. Place sample strips in two layers of plastic bag, press out air and place bag at room temperature. After 24 h, remove samples from the plastic bag. Fix the samples on forceps and immerse them in a 90 ° C water bath for 30 seconds and squeeze with a pad. Repeat soaking and squeeze twice. Rinse the fabric in cold tap water for at least 60 seconds and squeeze the water manually. Then air dry the tissue and measure TGEWA, residual pectin, wetting time and absorption test. The test result was shown in Table 2.

Example 4

Simultaneous degreasing and biolashing of Amylase Buffer Batch

Acid A and Pectinase Acid A

The same tissue and buffer system were used as Example 3. 100 ml of impregnation solution was added to each beaker and placed in the Lab-o-Mat, the solutions heated to 60 ° C. The beaker enzymes are removed and added according to Table 2 to the impregnation solution and mixed well. Fixed 2 sample strips of the same tissue on a pair of forceps. Immerse the sample strips in the soaking bath for 30 seconds and buffer with the pader. Repeated immersion and compression once again to ensure 100% moisture uptake. Place the sample strips in two layers of plastic bag, press out the air and place the bag in the preset water bath at 60 ° C. After 2 hours, the samples were removed from the plastic bag. Fixed the samples on the forceps and immerse them in a 90 ° C water bath for 30 seconds and squeezed with pader. Repeated soaking and squeezing twice. Rinse the fabric in cold spout water for at least 60 seconds and squeeze out the water manually. The tissue was then air dried and measured TEGEWA, residual pectin, wetting time and absorption test. The test result was shown in Table 2.

Table 2

<table> table see original document page 50 </column> </row> <table>

Example 5

Degumming and Biolavage of Batch Cold Buffer with Acid Amylase A and Acid Pectinase B

The procedures were the same as described in Example 3 except that Acid Pectinase B was used. The test result is shown in Table 3.

Example 6

Degumming and Biolavage of Acid Amylase Buffer and Acid B Pectinase Buffer

The procedures were the same as described in Example 4 except that Acid Pectinase B was used. The test result is shown in Table 3. Table 3

<table> table see original document page 51 </column> </row> <table>

Example 7

Simultaneous degreasing and biolava cold buffer flask with Amylase

Acid B and Pectinase Acid A

The procedures were the same as those described in Example 3 except that Acid Amylase A was replaced by Acid Amylase Β. The test result is shown in Table 4.

Example 8

Simultaneous degreasing and biolava Amylase Buffer Batch

Acid B and Pectinase Acid A

The procedures were the same as those described in Example 4, except that Acid Amylase A was replaced by Acid Amylase Β. The test result is shown in Table 4.

Table 4

<table> table see original document page 51 </column> </row> <table> Example 9

Simultaneous degreasing and biolavage Batch Cold buffer with Acid B Amylase and Acid B Pectinase

The procedures were the same as those described in Example 3, except that Acid Amylase A was replaced by Acid Amylase B and Acid Pectinase A was replaced by Acid Pectinase Β. The test result is shown in Table 6.

Example 10

Simultaneous degreasing and biolavage Batch Cold buffer with Acid B Amylase and Acid B Pectinase

The procedures were the same as those described in Example 4, except that Acid Amylase A was replaced by Acid Amylase B and Acid Pectinase A was replaced by Acid Pectinase Β. The test result is shown in Table 5.

Table 5

<table> table see original document page 52 </column> </row> <table>

Example 11

Wild-type Acid Alpha-Amylase A Cotton Fabric Degreasing

A 100% cotton fabric (270 g / m2) was from Borás WâfVeri Kingsfors AB, Sweden. It was produced in 2003 with Cupper 3/1 construction. The fabric contained 28 threads / cm of warp thread and 14 threads / cm of weft thread. The warp thread has Ne 11 and the warp thread has Ne 8. Both threads had the open end. The uptake of dry ironing on the warp thread was 8%. The ironing contained mainly Kollotex 5, Solvitose XO and beef tallow wax with emulsifier. Kollotex 5 is a low viscosity potato starch ester. Solvitose XO is a high viscosity starch ether with DS of about 0.07. The tissue sample strips were cut to about 25 g each.

The pH 3 buffer was produced by dissolving 11.53 g of 85% phosphoric acid in 4.5 liters of pure water, titrating with 5 N NaOH to pH 2.95, then adding 5 liters of water. . After adding 2 g / l of nonionic surfactant (a wetting agent) in the buffer, the pH of the buffer was measured as 3.05 at 25 ° C. The enzyme dose was added as listed in table 6.

The degumming treatment was conducted in a Lab-mat (Werner Mathis). A 250 ml buffer solution was added to each beaker. A given amount of alpha amylase enzyme was added. A tissue sample strip (25 g) was placed in each beaker. The beaker was closed and placed in Lab-o-mat. The beakers were heated to 50 ° C / min at 50 ° C by an infrared heating system fitted inside the Lab-mat. The beakers were spun at 30 rpm, 50 ° C for 45 minutes. After enzyme treatment, the tissue sample strip was sequentially washed with water in the same beaker three times at 95, 75 and 40 ° C, respectively.

After drying overnight in air, the tissue sample strip was stained with an iodine solution. The dyed tissue sample was visually compared to standard TGEWA photos at scale 1-9, where 1 is dark and 9 is colorless. Thus, the higher number indicates better starch removal. Visual assessment was performed by at least three professionals and an average TEGEWA value was provided for each tissue sample. The results are shown in Table 6. The residue of the metal ions on the fabric was also evaluated. The fabric was first cut through a 1 mm sieve with a Thomas-Wiley mill. Tissue paste 4.00 (± 0.01) g was mixed with 80 ml of 1 g / l EDTA solution. The mixture was incubated at 70 ° C and 200 rpm on a shaker (new Brunswick Scientific Co. Inc. Series 25) for 15 hours. After cooling for about 30 minutes, the mixture was centrifuged at 2500 rpm at 20 ° C for 10 minutes. The supernatant was collected for metal content analysis with a Perkinelmer atomic absorption spectrophotometer.

Table 6

<table> table see original document page 54 </column> </row> <table>

n / a = not measured.

Claims (32)

Process for the combined degreasing and washing of starch-containing starch fabric or starch derivatives during fabric manufacture, comprising incubating said starch fabric in an aqueous treatment solution having a pH in the range of 1 to 7 The aqueous treatment solution comprises an acid amylase and at least one acid wash enzyme. Process according to Claim 1, characterized in that said aqueous treatment solution has a pH in the range from 1 to 5, preferably from 1 to 4. Process according to Claim 1 or 2, characterized in that said washing enzyme is acid cellulase, acid pectinase, acid lipase, acid xylanase and / or acid protease or a mixture thereof. Process according to any one of claims 1 to 3, characterized in that the acid amylase is of bacterial or fungal origin, such as filamentous fungus origin. Process according to any one of claims 1 to 4, characterized in that the acid amylase is derived from a strain of Aspergilus, preferably Aspergillus niger, Aspergillus awamori, Aspergillus oryzae or Aspergillus kawachii, or a Rhizomucor strain, preferably Rhizomucor. pusillus, or a Meripilus strain, preferably a Meripilus giganteus strain. Process according to any one of claims 1 to 5, characterized in that an Aspergillus acid amylase is the Aspergillus niger acid alpha-amylase described in SEQ ID NO: 38, or a variant thereof. Process according to any one of claims 1 to 6, characterized in that the Rhizomucor acid amylase is Rhizomucor pusillus alpha-amylase described in SEQ ID NO: 48 or a variant thereof. Process according to any one of claims 1 to 7, characterized in that the acid amylase, preferably an acidic fungal alpha-amylase, is present in a concentration of 1-3,000 AFAU / kg of tissue, preferably 10 - 1,000 AFAU. / kg of tissue, especially 100-500 AFAU / kg of tissue or 1 - 3,000 AFAU / l of treatment solution, preferably 10-1,000 AFAU / l of treatment solution, especially 100-500 AFAU / l of treatment solution. Process according to any one of claims 1 to 8, characterized in that the bacterial acid amylase is derived from a strain of the genus Bacillus, preferably derived from a strain of Bacillus sp., More preferably a strain of Bacillus licheniformis, Bacillus. amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtllis, or Bacillus sp., such as Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM-9375, DSMZ 12648, DSMZ 12649, KSM K36, or KSM K38. Process according to any one of claims 1 to 9, characterized in that the alpha-amylase is the hybrid alpha-amylase shown in SEQ ID NO: 48, comprising a catalytic domain (CD) of Rhizomucor pusillus alpha-amylase. having an A. niger carbohydrate binding domain (CBD). Process according to claim 3, characterized in that said acid pectinase is an acid pectate lyase, an acid pectin lyase, an acid polygalacturonase and / or an acid polygalacturonate lyase. Process according to any one of claims 1 to 7, characterized in that said acid pectinase is Pectinex® BE XXL, Pectinex® BE Color, Pectinex® Ultra; Pectinex® Ultra SP-L, Pectinex® Yield Mash, Pectinex® XXL, Pectinex® Smash XXL, Pectinex® Smash, Pectinex ™ AR or any mixtures thereof. Process according to any one of claims 1 to 12, characterized in that said acid pectinase is derived from the genus Aspergillus or Bacillus. Process according to any one of claims 1 to 13, characterized in that said acid pectinase is added to or after addition of the acid amylase. Process according to any one of claims 1 to 14, characterized in that it is carried out at a temperature in the range of - 90 °, in particular 20 to 90 ° C. Process according to Claim 15, characterized in that the process is carried out at a temperature between 25 and 60 ° C for a suitable period of time, preferably between 2 and 24 hours. Process according to any one of claims 1 to 16, characterized in that the pH is in the range of pH 2 to 4. Process according to any one of Claims 1 to 17, characterized in that the fabric is made from natural or man-made fibers. Process according to any one of claims 1 to 18, characterized in that the fabric is woven of cotton, denim, linen, ramie, viscose, lyocel or cellulose acetate. Process according to any one of Claims 1 to 19, characterized in that the fabric is made of animal fibers, in particular silk or wool. A process according to any one of claims 1 to 20, characterized in that the fabric is made from man-made or naturally sourced polyester fibers such as poly (ethylene terephthalate) or poly (lactic acid). Process according to any one of Claims 1 to 21, characterized in that the fabric is made of nylon, acrylic or polyurethane fibers. Process according to any one of claims 1 to 22, characterized in that the fabric is a polyester-containing fabric or clothing consisting essentially of 100% polyester. Process according to any one of claims 1 to 23, characterized in that the polyester fabric is a polyester blend, such as a blend of polyester and cellulosic, including mixtures of polyester and cotton; a mixture of polyester and wool; a mixture of polyester and silk; a mixture of polyester and acrylic; a mixture of polyester and nylon; a polyester, blend of nylon and polyurethane; a mixture of polyester and polyurethane, rayon (viscose), cellulose acetate and tencel. Composition, characterized in that it comprises an acid amylase and an acid wash enzyme. Composition according to Claim 25, characterized in that the bacterial acid amylase is derived from a strain of the genus Bacillus, preferably derived from a strain of Bacillus sp, more preferably a strain of Bacillus licheniformis, Bacillus licheniformis, Bacillus amyloliquefaciens. Bacillus stearothermophilus, Bacillus subtilis, or Bacillus sp., Such as Bacillus sp. NCIB 12289, NCIB-12512, NCIB 12513, DSM 9375, DSMZ 12648, DSMZ 12649, KSM AP1378, KSM K36 or KSM K38. Composition according to Claim 25 or 26, characterized in that said acid amylase is derived from Aspergillus niger or Rhizomucor pusillus or mixtures thereof. Composition according to Claim 25 or 26, characterized in that said acid wash enzyme is selected from the group consisting of acid cellulase, acid pectinase, acid lipase, acid xylanase and / or acid protease, and mixtures thereof. Composition according to any one of Claims 25 to 28, characterized in that said acid pectinase is derived from a strain of Aspergillus or Bacillus. Composition according to any one of Claims 25 to 29, characterized in that said acid pectinase is Pectinex® BE XXL, Pectinex® BE Color5 Pectinex® Ultra; Pectinex ™ Uitra SP-L, Pectinex® Yield Mash, Pectinex® XXL, Pectinex® Smash XXL, Pectinex® Smash and / or Pectinex ™ AR. Composition according to any one of Claims 25 to 30, characterized in that said composition further comprises stabilizer, surfactant, wetting agent, dispersing agents, sequestering agents and emulsifying agents or a mixture thereof. Use of a composition as defined in any one of claims 25 to 31, characterized in that it is for simultaneous degreasing and washing.